**5. Possible redox regulations of KATP and other channels**

The structure of KATP has been resolved and numerous mutagenesis studies of KATP have been conducted. Amino acid residues that are candidate redox targets are yet to be identified. The KATP channel is a hetero-octamer consisting of four external regulatory sulfonylurea receptor 1 (SUR1, a product of *Abcc8* gene) subunits and four pore-forming subunits of potassium inward rectifier, Kir6.2 (*Kcnj11* gene) [88, 89]. These Kir6.2 subunits cluster in the middle of ~18 nm size structure with a ~13 nm height [90]. The part exposed to the cytosol contains an ATP binding site, located about 2 nm below the membrane. A single ATP molecule was reported to close the channel, i.e. with the other three binding sites left unoccupied [91]. However, the ATP binding site overlaps with the binding site for phosphatidylinositol 4,5-bisphosphate (PIP2), which stabilizes the open state. Palmitoylation of Cys166 of Kir6.2 was then reported to amplify the responsiveness to PIP2 [92]. Upon the release of PIP2 from the binding site, the open probability becomes decreased [90, 93, 94].

Diazoxide or cromakalim, as well as numerous other openers, set KATP pharmacologically in the open state even at a high ATP concentration [95]. In contrast, the artificial KATP closing by sulfonylurea derivatives, such as glibenclamide, takes place independently of ATP. Besides this sulfonylurea binding site, each of the four SUR1 subunits contains MgATP and MgADP binding sites. MgATP is hydrolyzed at the nucleotide binding fold 1 (NBF1) to MgADP. Resulting MgADP subsequently activates KATP at NBF2. This is indeed reflected by the ATP-sensitive increase in K<sup>+</sup> conductance and following lower excitability, accompanied by the lower sensitivity to ATP inhibition [91].

The phosphorylation of KATP reportedly sets the sensitivity of the KATP ensemble. The setting is such that transitions upon the glucose rise from 3 or 5 mM to 7 mM or > 10 mM result in the closing of the remaining 10% of the initially open channels by elevations between just the two ATP concentrations falling into the mM range. Any redox component in this was never indicated and should be studied. Nevertheless, phosphorylation mediated by the protein kinase A (PKA) was already reported to act in this unusual setting. Thus Thr224 [96] and Ser372 were reported to be the verified PKA phosphorylation sites. Their phosphorylation increases the open probability of KATP [97]. This might hypothetically provide closing mechanism acting at higher ATP concentration or even requiring H2O2. In a longer time scale, phosphorylation also increases the number of channels in the plasma membrane. Also, Thr224 was found to be phosphorylated by Ca2+ and calmodulin-dependent kinase II (CaMKII) while interacting with βIV-spectrin [98]. In vivo, also autonomic innervations and paracrine stimulation ensure sufficient PKA-mediated phosphorylation of KATP.

Since the original discovery of the essential role of KATP in GSIS [99], only an indirect inhibition of KATP by H2O2 was observed in smooth muscle cells [100]. Nevertheless, other redox-sensitive targets have been identified in pancreatic β-cells. But we can exclude the possibility that the IGV exocytosis itself might be directly induced by H2O2, independently of Ca2+ [52], since the ability of exogenous H2O2 to induce insulin secretion in INS-1E cells was only partially blocked by NOX4-siRNA, but it was completely blocked by a CaL blocker nimodipine [1]. Consequently, albeit the used H2O2 doses exceeded 100 μM, they did not directly stimulate the KATP-independent exocytosis of insulin granules.

A second possibility would be that CaL channels themselves may be hypothetically co-activated by H2O2. Third, the plasma membrane depolarization might be redox sensitive, so that H2O2 could directly or indirectly inhibit repolarizing K+ -channels, such as KV [101–103]. The fourth plausible redox link with GSIS would concern with the reported redox activation of TPRM2 depolarizing channels [2]. The latter is the most plausible, since it is related to a Ca2+-induced [52, 104] or H2O2-induced exocytosis of insulin granules by the H2O2-activation of TPRM2 depolarizing channels [2, 105]. Note, our results excluded the Ca2+-independent H2O2-induced exocytosis of IGVs at least in rat pancreatic β-cells [1]. Therefore, if the H2O2-activated TRPM2-dependent mechanism exists, it must provide the required synergy with KATP, to reach the −50 mV plasma membrane depolarization threshold. Note also, that TRPM2 was already implicated as a significant player in the GLP-1 potentiation of insulin secretion [106].

Finally, a competition for NADPH between NOX4 and a hypothetical NADPHactivated K<sup>+</sup> -channel could exist. Nevertheless, using patch-clamped INS-1E cells in a whole cell mode, we demonstrated a closure of KATP by H2O2 produced by NOX4 at high glucose, since in cells silenced for NOX4, even ATP resulting from the metabolism of high glucose was unable to close the KATP channel [1].

#### **6. Receptor-mediated amplification of insulin secretion**

G protein-coupled receptors activating heterotrimeric G proteins ensure pleiades of cell responses, mutually interrelated. G proteins typically regulate production of second messengers. Thus Gαs proteins increase generation of cyclic AMP (cAMP), whereas Gαi/o proteins decrease it [107–109]. The G proteins Gαq/11 initiate PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DAG) and IP3 [110, 111]. Gα12/13 proteins promote protein RhoA

**41**

*Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

G protein classes rather control long-term effects.

proteins directly activated by cAMP 2 (EPAC2) [117–119].

hence exocytosis of insulin granules [122].

SNAP-25 [124].

In pancreatic β-cells, the PKA pathway is involved in signaling of incretin (GLP-1 and GIP) receptors [107, 120]. It exerts a minor contribution to signaling from metabotropic receptors, such as GPR40, which is sensing long chain fatty acids [111]. PKA typically amplifies the Ca2+-dependent exocytosis of insulin granules. The core pathway involves PKA phosphorylation and hence activation of the CaL β2-subunit, in concert with KATP phosphorylation decreasing the ATP concentration range required for its closure (see above) [121]. In addition, PKA inhibits Kv channels, which otherwise terminate plasma membrane depolarization; hence this prolongs already more intensive Ca2+ influx via phosphorylated CaL and

Another PKA target is the exocytosis-modulating protein termed snapin, the phosphorylation of which allows its interaction with the other IGV proteins, which enhances the 1st GSIS phase [123]. Snapin participates in tethering of IGVs to the plasma membrane by coiled-coil interaction with a lipid-anchored protein

Altogether, the PKA pathway ensures about 50% of cAMP responses in β-cells [125], while the EPAC2 pathway ensures the remaining responses [117–119]. EPAC2 protein possesses a guanine nucleotide exchange activity, thus inducing the Ca2+ induced Ca2+release from ER via RyR [126] (questioned in [127]), occurring only at high glucose, since it requires the primary CaL opening [128], which also partially refills the ER Ca2+ stores. The EPAC2 pathway also affects the IGV proteins and thus facilitates the insulin exocytosis. For example, Rim2a protein is a target [129, 130], located on the inner plasma membrane surface and on IGVs, representing a scaffold for IGV exocytosis [131]. Rim2a interacts with Rab3A of IGVs and the resulting Rim2a-Rab3A complex facilitates docking of IGVs into the plasma membrane. This is followed by so-called priming, which is subsequently initiated by the Rim2a interaction with the Munc13–1 protein. Munc13–1 then opens syntaxin 1 from its closed conformation, thus allowing fusion with the plasma membrane. EPAC2 also interacts with NBD1 of SUR1, being released by cAMP [35]. Such locally released EPAC2 induces the release of Rim2 from the α1.2 CaL subunit.

for remodeling of the cytoskeleton [112]. Class of proteins termed β-arrestins initiates signaling via proximal MAP kinase, IκB, and Akt pathways [113]. The latter two

Let us emphasize downstream pathways that are important for acute effects in pancreatic β-cells, which predominantly lead to either modulation of the plasma membrane channels, typically CaL, KATP and KV, so to ensure more intensive insulin secretion; or their action evokes stimulation of insulin secretion via ensuring the surplus Ca2+ influx to the cytosol from ER or mitochondria; or, else, their action targets proteins of the exocytotic machinery on the IGV or plasma membranes. The latter responses alter the kinetics of IGVs in docking, priming and fusion with the plasma membrane, so to facilitate exocytosis. Interestingly, these events could be independent of CaL and theoretically could take place at low glucose concentrations. Activation of Gαs increases the activity of transmembrane adenylate cyclases (tmAC) producing cAMP from ATP [108, 109]. A number of phosphodiesterases (of 11 families) degrade cAMP (some also or exclusively cGMP). cAMP is a universal 2nd messenger having a specific function in amplifying of GSIS and insulin secretion stimulated with other secretagogues. Also, soluble adenylate cyclases (sACs) exist, notably in the mitochondrial matrix, while their reaction is potentiated by Ca2+ and bicarbonate. The major mediators of cAMP effects are cAMP-dependent PKA [114], including PKA tethered to the outer mitochondrial membrane [115, 116], and the parallel pathway of enhanced signaling via exchange

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

phosphorylation of KATP.

The phosphorylation of KATP reportedly sets the sensitivity of the KATP ensemble. The setting is such that transitions upon the glucose rise from 3 or 5 mM to 7 mM or > 10 mM result in the closing of the remaining 10% of the initially open channels by elevations between just the two ATP concentrations falling into the mM range. Any redox component in this was never indicated and should be studied. Nevertheless, phosphorylation mediated by the protein kinase A (PKA) was already reported to act in this unusual setting. Thus Thr224 [96] and Ser372 were reported to be the verified PKA phosphorylation sites. Their phosphorylation increases the open probability of KATP [97]. This might hypothetically provide closing mechanism acting at higher ATP concentration or even requiring H2O2. In a longer time scale, phosphorylation also increases the number of channels in the plasma membrane. Also, Thr224 was found to be phosphorylated by Ca2+ and calmodulin-dependent kinase II (CaMKII) while interacting with βIV-spectrin [98]. In vivo, also autonomic innervations and paracrine stimulation ensure sufficient PKA-mediated

Since the original discovery of the essential role of KATP in GSIS [99], only an indirect inhibition of KATP by H2O2 was observed in smooth muscle cells [100]. Nevertheless, other redox-sensitive targets have been identified in pancreatic β-cells. But we can exclude the possibility that the IGV exocytosis itself might be directly induced by H2O2, independently of Ca2+ [52], since the ability of exogenous H2O2 to induce insulin secretion in INS-1E cells was only partially blocked by NOX4-siRNA, but it was completely blocked by a CaL blocker nimodipine [1]. Consequently, albeit the used H2O2 doses exceeded 100 μM, they did not directly

A second possibility would be that CaL channels themselves may be hypothetically co-activated by H2O2. Third, the plasma membrane depolarization might be redox sensitive, so that H2O2 could directly or indirectly inhibit repolarizing

Finally, a competition for NADPH between NOX4 and a hypothetical NADPH-

a whole cell mode, we demonstrated a closure of KATP by H2O2 produced by NOX4 at high glucose, since in cells silenced for NOX4, even ATP resulting from the metabo-

G protein-coupled receptors activating heterotrimeric G proteins ensure pleiades of cell responses, mutually interrelated. G proteins typically regulate production of second messengers. Thus Gαs proteins increase generation of cyclic AMP (cAMP), whereas Gαi/o proteins decrease it [107–109]. The G proteins Gαq/11 initiate PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DAG) and IP3 [110, 111]. Gα12/13 proteins promote protein RhoA



stimulate the KATP-independent exocytosis of insulin granules.

the GLP-1 potentiation of insulin secretion [106].

lism of high glucose was unable to close the KATP channel [1].

**6. Receptor-mediated amplification of insulin secretion**

**40**

K+

activated K<sup>+</sup>

for remodeling of the cytoskeleton [112]. Class of proteins termed β-arrestins initiates signaling via proximal MAP kinase, IκB, and Akt pathways [113]. The latter two G protein classes rather control long-term effects.

Let us emphasize downstream pathways that are important for acute effects in pancreatic β-cells, which predominantly lead to either modulation of the plasma membrane channels, typically CaL, KATP and KV, so to ensure more intensive insulin secretion; or their action evokes stimulation of insulin secretion via ensuring the surplus Ca2+ influx to the cytosol from ER or mitochondria; or, else, their action targets proteins of the exocytotic machinery on the IGV or plasma membranes. The latter responses alter the kinetics of IGVs in docking, priming and fusion with the plasma membrane, so to facilitate exocytosis. Interestingly, these events could be independent of CaL and theoretically could take place at low glucose concentrations.

Activation of Gαs increases the activity of transmembrane adenylate cyclases (tmAC) producing cAMP from ATP [108, 109]. A number of phosphodiesterases (of 11 families) degrade cAMP (some also or exclusively cGMP). cAMP is a universal 2nd messenger having a specific function in amplifying of GSIS and insulin secretion stimulated with other secretagogues. Also, soluble adenylate cyclases (sACs) exist, notably in the mitochondrial matrix, while their reaction is potentiated by Ca2+ and bicarbonate. The major mediators of cAMP effects are cAMP-dependent PKA [114], including PKA tethered to the outer mitochondrial membrane [115, 116], and the parallel pathway of enhanced signaling via exchange proteins directly activated by cAMP 2 (EPAC2) [117–119].

In pancreatic β-cells, the PKA pathway is involved in signaling of incretin (GLP-1 and GIP) receptors [107, 120]. It exerts a minor contribution to signaling from metabotropic receptors, such as GPR40, which is sensing long chain fatty acids [111]. PKA typically amplifies the Ca2+-dependent exocytosis of insulin granules. The core pathway involves PKA phosphorylation and hence activation of the CaL β2-subunit, in concert with KATP phosphorylation decreasing the ATP concentration range required for its closure (see above) [121]. In addition, PKA inhibits Kv channels, which otherwise terminate plasma membrane depolarization; hence this prolongs already more intensive Ca2+ influx via phosphorylated CaL and hence exocytosis of insulin granules [122].

Another PKA target is the exocytosis-modulating protein termed snapin, the phosphorylation of which allows its interaction with the other IGV proteins, which enhances the 1st GSIS phase [123]. Snapin participates in tethering of IGVs to the plasma membrane by coiled-coil interaction with a lipid-anchored protein SNAP-25 [124].

Altogether, the PKA pathway ensures about 50% of cAMP responses in β-cells [125], while the EPAC2 pathway ensures the remaining responses [117–119]. EPAC2 protein possesses a guanine nucleotide exchange activity, thus inducing the Ca2+ induced Ca2+release from ER via RyR [126] (questioned in [127]), occurring only at high glucose, since it requires the primary CaL opening [128], which also partially refills the ER Ca2+ stores. The EPAC2 pathway also affects the IGV proteins and thus facilitates the insulin exocytosis. For example, Rim2a protein is a target [129, 130], located on the inner plasma membrane surface and on IGVs, representing a scaffold for IGV exocytosis [131]. Rim2a interacts with Rab3A of IGVs and the resulting Rim2a-Rab3A complex facilitates docking of IGVs into the plasma membrane. This is followed by so-called priming, which is subsequently initiated by the Rim2a interaction with the Munc13–1 protein. Munc13–1 then opens syntaxin 1 from its closed conformation, thus allowing fusion with the plasma membrane. EPAC2 also interacts with NBD1 of SUR1, being released by cAMP [35]. Such locally released EPAC2 induces the release of Rim2 from the α1.2 CaL subunit.

The local Ca2+ influx within CaL ensures EPAC2 binding to Rim2, and subsequent interaction with another Ca2+ sensor termed Piccolo. The heterotrimeric complex then interacts with Rab3A and enables IGV exocytosis.

Interestingly, all necessary components of the PKA pathway were identified in the mitochondrial matrix, including sAC, PDE2A2 [132], and also PKA [133]. However, we may also speculate that some proteins can be phosphorylated by cytosolic PKA or by its fraction attached to OMM prior to their import to the mitochondrial matrix. There was also a consensus that cAMP cannot freely diffuse to the matrix [132]. Thus cAMP in the mitochondrial matrix may act as an independent pool [134, 135]. Its source is the matrix-located soluble adenylate cyclase sAC, which is activated by bicarbonate and Ca2+ [136, 137]. Since CO2 is increasingly released when the Krebs cycle turnover increases upon GSIS, the matrix localized mtPKA can be activated in this way [138]. In any case, OXPHOS is facilitated in mitochondria of numerous tissues via phosphorylation of Complex I NDUFS4 subunit (facilitating its Hsp70-mediated import), Complex IV COXIV-1 subunit (preventing its inhibition by ATP) [139] as well as via IF1, enhancing ATP synthesis by disabling the inhibitory binding of phosphorylated IF1 dimers to the ATP synthase [140]. A link to redox homeostasis can be viewed in the observed release of the PKA catalytic subunits by the increased ROS [141, 142]. Thus mtPKA can act in parallel to the cytosolic PKA signaling initiated by GPR40 and GLPR or GIPR receptors. PKA targeting of at least IF1, and probably also of Complex I and Complex IV, should contribute to the amplification of insulin secretion by FAs or incretins.

The G protein Gαq/11 initiates signaling through the phospholipase C (PLC-) mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate into DAG and inositol triphosphate IP3 [110]. The main effector of DAG is protein kinase C (PKC), which is activated by DAG. One of the effectors of IP3 is the IP3 receptor (IP3R; subtypes IP3R1, IP3R2 and IP3R3), which is another important Ca2+ channel residing on ER membranes in β-cells [143]. Similarly to the EPAC2-RyR route of Ca2+ release from ER Ca2+, the opening of this channel amplifies the primary CaL mediated Ca2+ signaling for insulin release. PKC contributes to the plasma membrane depolarization, while activating TRPM4 and TRPM5 [144]. Besides the canonical plasma membrane effects, PKC and downstream ERK1/2 signaling stimulates OXPHOS, hence mitochondrial ATP synthesis [145].
